EGFP-Cre重组酶真核表达重组质粒的构建及表达

Construction of EGFP-Cre Recombinase into Eukaryotic Recombinant Plasmid

目的构建噬菌体P1Cre重组酶真核表达重组质粒,检测其在体外的表达情况和生物学活性。方法利用分子克隆技术,将cre编码序列克隆到真核表达质粒pIRES2-EGFP上,构建为重组质粒pCMV-Cre-EGFP。行菌液PCR、BamHⅠ酶切及测序鉴定,用FuGENE6介导转染293T细胞,应用荧光显微镜和流式细胞仪观察分析Cre重组酶的表达和重组效应。结果经菌液PCR、酶切及测序等鉴定分析,证实已将目的片段cre插入pIRES2-EGFP的多克隆位点上;建立Cre(+)组(pCMV-Cre-EGFP和含Loxp-CMV-RFP-Loxp的pCSilencer质粒)和Cre(-)组(pIRES2-EGFP和pCSilencer),转染293T细胞,48h后荧光显微镜下可见2组均表达绿色荧光和红色荧光,Cre(+)组的红色荧光少于Cre(-)组;流式细胞仪检测荧光蛋白表达率,统计学分析显示2组细胞的红色荧光蛋白表达率存在显著性差异(P<0.01)。结论成功构建噬菌体P1Cre重组酶表达重组质粒,并能在体外表达具有活性的Cre重组酶。

Objective To construct and identify recombinase Cre eukaryotic expression vector. Methods The cre insert was cloned into the eukaryotic expression vector plRES2-EGFP. The recombinant plasmid was identified by colony PCR analysis, restriction enzymolysis（BamH Ⅰ ）, and DNA sequencing. After transfecting 293T cells mediated by FuGENE 6, the expression and recombination effect of Cre were analyzed. Results The cre gene was cloned into the multiple cloning site of the vector successfully. pCMV-Cre-EGFP and pCSilencer（which contains a part of Loxp-CMV-RFP-Loxp） were cotransfected with 293T cells in Cre （＋） group, plRES2-EGFP and pCSilencer were cotransfected in Cre （-） group. 48 h later, cells of both groups were visualized for EGFP or RFP by fluorescence microscopy and examined with flow cytometry. group（P 〈 0. 01 ）. Conclusion successfully. The expression of the RFP in Cre（＋） group was less than in Cre（-） pCMV-Cre-EGFP eukaryotic expression plasmid has been constructed And it can express active recombinase Cre in vitro.